site-specificity in intermediate fi'lament-membrane interactions

Transcription

1 Pro. Natl. Aad. Si. USA Vol. 84, pp , Otober 1987 ell Biology Binding of two desmin derivatives to the plasma membrane and the nulear envelope of avian erythroytes: videne for a onserved site-speifiity in intermediate fi'lament-membrane interations (lamin B/ankyrin/membrane skeleton/karyoskeleton) SPYROS D. GORGATOS*, KLAUS WBRt, NORBRT GSLRt, AND GUNTR BLOBL* *Laboratory of ell Biology, Howard Hughes Medial nstitute, The Rokefeller University, New York, NY 121; and tmax Plank nstitute for Biophysial hemistry, D-34 Goettingen, Federal Republi of Germany ontributed by Gunter Blobel, June 9, 1987 ABSTRAT Using solution binding assays, we found that a 45-kDa fragment of desmin, laking 67 residues from the N terminus, ould speifially assoiate with avian erythroyte nulear envelopes but not with plasma membranes from the same ells. t was also observed that a 5-kDa desmin peptide, missing 27 -terminal residues, retained the ability to bind to both membrane preparations. Displaement experiments with an exess of purified vimentin suggested that the two desmin derivatives were interating with a previously identified vimentin reeptor at the nulear envelope, the protein lamin B [Georgatos, S. & Blobel, G. (1987) J. eu Biol. 15, ]. Additional analysis by affinity hromatography onfirmed this onlusion. mploying an overlay assay, we demonstrated that the 5-kDa fragment, but not the 45-kDa desmin peptide, was apable of interating with the plasma membrane polypeptide ankyrin (a known vimentin attahment site), as was intat vimentin. onversely, the nulear envelope protein lamin B was reognized by both fragments but not by a hymotrypti peptide omposed solely of the helial rod domain of desmin. These data imply that the lamin B-binding site on desmin resides within the 21 residues following its helial rod domain, whereas the ankyrin-assoiating region is loalized within its N-terminal head domain, exatly as in the ase of vimentin. arlier ultrastrutural observations have revealed a lose topologial assoiation of intermediate filaments (F) with the nulear envelope and the plasma membrane in several model systems (1-6). n line with these data, reent biohemial studies have suggested that at least vimentin F may be anhored to the subplasmalemmal membrane-skeleton and the karyoskeleton via speifi, proteinaeous linkers. Two types of suh high-affinity vimentin "reeptors" have been identified in avian and mammalian erythroytes: the peripheral plasma membrane protein ankyrin, whih also onnets the spetrin-atin meshwork to the plasma membrane proper (7-1), and the polypeptide lamin B (11), a onstituent of the fibrous nulear lamina that lines the nuleoplasmi side of the inner nulear membrane (12). Vimentin, as well as all other F subunits known so far, possesses a tripartite substruture. t onsists of a entral helial domain flanked by two nonhelial segments at the N- and -terminal regions, the so-alled "head" and "tail" domains (13). These peripheral domains exhibit distintly different properties in terms of membrane binding: the N-terminal head domain of vimentin binds to ankyrin, while its -terminal tail region binds to lamin B. Beause the head domain seems also to be involved in filament formation (14, 15), ankyrin, by binding to it, ould behave as a filament "apping" fator bloking F elongation at the assoiation The publiation osts of this artile were defrayed in part by page harge payment. This artile must therefore be hereby marked "advertisement" in aordane with 18 U.S solely to indiate this fat. sites. Lamin B, on the other hand, seems to promote filament nuleation by assoiating in a ooperative fashion with the tail domain. The entral domain does not appear to be involved in any of these interations. On the basis of the above information, a funtional polarization during F assembly in vivo has been postulated (11), in spite of the apparent strutural apolarity of 1-nm filaments reonstituted in vitro from isolated subunits (16, 17). To test the general validity of this hypothesis, we deided to examine the binding properties of a different F subunit, desmin, taking advantage of the strutural and funtional similarities between desmin and vimentin (18, 19) and exploiting two desmin fragments that had been previously haraterized (15). Here, we provide evidene that desmin and vimentin ould share the same attahment sites. Sine we also detet a signifiant affinity differene in the binding of desmin versus vimentin to avian erythroyte lamin B, we predit the existene in musle ells of a lamin B isotype that may be more finely tailored for onneting the desmin skeleton to the nuleus. MATRALS AND MTHODS Membranes and Probes. Nulear envelopes and plasma membranes from turkey erythroytes (depleted or not depleted of vimentin) were isolated as desribed (11), exept that the envelopes were salt-washed with 1 M K/2 mm sodium phosphate/4 mm DTA/1 mm dithiothreitol/.2 mm phenylmethylsulfonyl fluoride, ph 8., for 15 min on ie, to enrih the preparation in nonhistone polypeptides. alf lens vimentin, turkey erythroyte lamins, and rat liver lamins were purified as desribed (11). The two hiken desmin derivatives employed in this study (T-desmin and L-desmin, derived by digestion with thrombin and endoproteinase Lys-, respetively) were isolated as speified (15), whereas the helial rod domain of desmin was obtained from one of the derivatives (the thrombi fragment) after brief treatment with hymotrypsin followed by ion-exhange hromatography (13). The purified proteins were 1251-labeled under mild onditions with Bolton-Hunter reagent (1). Anti-ankyrin antibodies (prepared against human erythroyte ankyrin) as well as anti-spetrin antibodies were a generous gift of V. Marhesi (Yale University); the haraterization of the anti-lamin B antibodies has been reported (11, 2). Anti-lamin A and antibodies were kindly provided by L. Gerae (Johns Hopkins University). Abbreviations: F, intermediate filament(s); T-desmin, thrombi fragment of desmin; L-desmin, fragment of desmin obtained by digestion with the lysine-speifi endoproteinase Lys-. 678

2 2 -o,1 -o D. ) A_ M ell Biology: Georgatos et al. 8 4 HAD * MDDL DOMAN * TAL A * L-DSMN JO- Lys 1 nulear envelopes /,~~~~~~ n plasma membranes ~ B Pro. Natl. Aad. Si. USA 84 (1987) 6781 Assays. Solution binding assays with iodinated proteins and isolated membrane frations were performed as previously (11), exept that the assay buffer ontained 1 mm Tris Hl (ph 7.3), 15 mm Na, 2 mm Mg" l2, 1 mm dithiothreitol,.1 mm phenylmethylsulfonyl fluoride, and.1 mg of bovine serum albumin per ml, unless stated otherwise. All data points in Fig. 1 are averages of at least four independent determinations. n Fig. 1 only the speifi binding is shown; the nonspeifi binding was measured using proteolyzed membranes and urea-extrated envelopes as in ref. 11 and amounted to about 2o of the total. The quantities of radiolabeled L- and T-desmin that were pelleted in the absene of membranes were about 2 orders of magnitude less than the speifi binding. Affinity hromatography was onduted exatly as desribed (11), using a lamin B-agarose olumn. Overlay binding assays were exeuted as follows. Membrane preparations or protein frations were solubilized in Laemmli sample buffer (21) that also ontained 6 M urea, without boiling. Samples were eletrophoretially frationated in 1% polyarylamide gels (separating gel, 12 m), run at 9 ma. The proteins were eletrophoretially transferred to nitroellulose filters (5 V, 5 hr, 23) in a buffer ontaining 4 g ofnadodso4, g ofglyine, 12.1 g of Tris (base), and 8 ml of methanol per 4 liters. After transfer, the filters were washed with 5%6 2-propanol in water (5-1 min, 1 ml per strip), rinsed extensively with distilled water, and washed for 1 hr with 1mM Tris Hl/15 mm Na/.1% Tween 2, ph 7.3. To renature the proteins, the filters were then treated for a minimum of 18 hr with buffer A (15 mm Tris Hl/15 mm Na/2 mm Mgl2/1 mm dithiothreitol,.1 mm phenylmethylsulfonyl fluoride/.1% Tween 2/.1% gelatin, ph 7.3, at 23). At the end of this inubation, the filters were bathed for 1 hr in fresh buffer and then inubated with buffer A ontaining the appropriate traers as speified in the figure legends (3 hr, 23). Finally, the filters were washed six times with =9 ml of buffer A over a 2-hr period. After drying, they were autoradiographed on Kodak XAR film for 3-15 hr at -7. Other Proedures. letrophoresis was aording to Laemmli (21) and protein determinations were made aording to Lowry et al. (22) and Fenner et al. (23). RSULTS Quantitative Aspets of Binding. Previous studies (15) had established that upon prolonged thrombi digestion, the F protein desmin (Mr 53,) ould be onverted into a smaller derivative (T-desmin) that laks the ability to polymerize and ontains only the helial middle domain and the -terminal tail of the original moleule. Under isotoni salt onditions and at neutral ph this preparation is fairly homogeneous and onsists of tetramers with subunit Mr 45,. To assess the membrane-binding ativity of this fragment, 1251-labeled T- desmin was ombined with plasma membranes or nulear envelopes depleted of endogenous F (eletrophoreti profiles shown in Fig. 1) and the binding was measured by a sedimentation assay (11). When T-desmin was oinubated with turkey erythroyte plasma membranes, no signifiant speifi binding was deteted (Fig. 1B, lower urve). However, when nulear HAD * MDDL DOMAN *TAL TH- ATHY T-DSMN ia nulear " envelopes a ' X' pg/m Free 25 -L- Desmin if K,, plosmo membranes plg /m Free 125 -T- Desmin B L) a) Q LDO D *- D3 a) _ f mss ). 4 8 bound 125 -L- Desmin bound 125-T-Desmin FG. 1. Binding of hiken desmin fragments to plasma membranes and nulear envelopes from turkey erythroytes. (A) Binding of 1251-labeled L-desmin to plasma membranes (e) or nulear envelopes (o). The assay was exeuted as speified in Materials and Methods with iodinated desmin fragments at final speifi ativities of 2-55 pm/bg, at 5 mm Nal. (B) As in A, but with '251-labeled T-desmin (speifi ativity 4 pm/flg) at 15 mm Na. n both ases the nonspeifi binding was subtrated. () letrophoreti profiles of plasma membranes (lane 1), nulear envelopes (lane 2), T-desmin (lane 3), L-desmin (lane 4), vimentin (lane 5), rat liver lamins A and (lane 6), and rat liver lamin B (lane 7) used in the assays. Proteins were visualized by oomassie blue staining. (D and ) Sathard plots of the binding of L-desmin and T-desmin (respetively) to nulear envelopes. The shemes in A and B represent the strutural features of the two desmin derivatives. The site (Lys) where desmin is leaved by the lysine-speifi protease to produe L-desmin and the thrombin site (TH) along the desmin moleule where leavage generates T-desmin are indiated. The major strutural domains (head, middle, tail) of desmin are also indiated.

3 6782 ell Biology: Georgatos et al. envelopes from the same ells were substituted for the plasma membranes, a signifiant quantity of the traer was found to partition with the membrane pellets. Quantitative assessment of the binding revealed a onentration-dependent, saturable assoiation (Fig. 1B, upper urve). n essene, T-desmin appeared to behave like some vimentin peptides that lak parts of or the entire head domain and thereby are inompetent to polymerize or assoiate with the ankyrin moleule (1, 14). On the other hand, T-desmin shared with a 6.6-kDa vimentin fragment (ontaining exlusively its tail domain) the ability to bind to nulear envelopes in a saturable manner (11). Beause these results were onsistent with the notion that desmin subunits, like vimentin subunits, may utilize opposite end-domains to interat with the nulear envelope and the plasma membrane, we used another derivative of desmin in order to map the ative sites along the desmin moleule more preisely, A proteolyti produt was prepared by digesting isolated desmin with a lysine-speifi protease, yielding L-desmin, whih ontains an intat N-terminal domain, the entire middle domain, and part of the tail domain, missing only 27 residues from the -terminus. L-desmin is polymerization-ompetent and behaves like intat desmin under hypotoni salt onditions (5 mm salt). At physiologial ioni strength it an form bundled filaments (15). When binding was assessed using radiolabeled L-desmin (at 5 mm Nal), a saturable assoiation with the plasma membranes was observed (Fig. LA, lower urve). The same probe bound also to nulear envelopes in a onentrationdependent manner (Fig. 1A, upper urve). Despite the qualitative similarities in the behavior of desmin and vimentin, there were some noteworthy differenes in their binding to the nulear envelope. Both desmin derivatives bound to the nulear envelopes with a lower affinity than vimentin. This was partiularly evident after Sathard analysis of the data (Fig. 1D and ), whereby assoiation onstants of approximately 2 x 16 M-1 and 1.2 x 16 MW1 were dedued for L-desmin and T-desmin, respetively. Analogous measurements with vimentin, or its -terminal tail domain, have yielded values ranging from 17 to 2.1 x 17 M-1 (11). dential values were obtained upon repetition of vimentin binding assays with the salt-washed envelope preparations used in this study (data not shown). Thus, it appeared that the desmin-envelope assoiation was substantially weaker than the vimentin-envelope interation. 1 Another differene in the quantitative features of the binding onerned the issue of ooperativity: in previous experiments it was shown that the primary interation of the vimentin tail with its nulear binding site was haraterized by a pronouned positive ooperativity (11). This was not observed in the ase of desmin, as evidened by the shape of the Sathard plots (Fig. 1D and ). dentifliation of Binding Sites. To investigate the nature of the desmin binding sites at the nulear envelope, as well as their relationship to the vimentin reeptor, various amounts of unlabeled vimentin were oinubated with the iodinated desmin fragments and nulear envelopes, and the displaement of the traers was quantitated. As seen in Fig. 2A, vimentin was apable of inhibiting most of the T-desmin binding (the same happened with L-desmin, although higher amounts of unlabeled vimentin were required to ahieve equivalent displaement; data not shown). These data suggested that the desmin derivatives and vimentin were binding to the same reeptor sites at the nulear envelope. Nevertheless, when Dixon analysis (24) was attempted, to hek whether the inhibition we had observed was of a purely ompetitive mode, it was realized that, apart from a diret vimentin/desmin ompetition for the same site, the two proteins must have reated also with eah other (Fig. 2B). This was not unexpeted, sine independent studies (25) had shown that desmin and vimentin ould opolymerize in vitro into heteropolymeri forms. However, the unertainty assoiated with these interpretations ditated additional analysis in order to demonstrate diret interations. A lamin B-agarose olumn was used to examine whether the desmin peptides, like Pro. Natl. Aad. Si. USA 84 (1987) vimentin, ould bind diretly to lamin B. Both T- and Udesmin were quantitatively retained by the olumn and subsequently eluted with 7 M urea (Fig. 2). To onfirm these results we deided to develop an overlay assay for examining the diret assoiations of both desmin and vimentin with their putative membrane reeptors. Highspeifi-ativity 125-labeled T-desmin, L-desmin, vimentin, or the desmin middle domain (obtained after leaving T- desmin with hymotrypsin) were prepared and used to probe blotted eletrophorograms of plasma membranes and nulear envelopes that ontained some endogenous vimentin serving as an internal marker. As shown in Fig. 3 (lanes ), L-desmin, T-desmin, and intat exogenous vimentin bound to lamin B and to endogenous vimentin ontained in the nulear enve- B - w -Qo le VN 'D -o 3 F Vimentin added ( t/aossay) FG. 2. (A) Displaement of 1251-labeled T-desmin from nulear envelopes by an exess of unlabeled vimentin. The assay mixture (1 Al) ontained.2,ug of -labeled T-desmin (speifi ativity 8, pm/;ug) and 15,ug of nulear envelopes. Vimentin was added as a 15-;&g/ml solution. (B) Two assays similar to the one shown in A, with 'n-labeled T-desmin at 1.5 "$g/ml (e) or 4. ;ug/ml (o). Data are plotted aording to Dixon (24). () Binding of the two desmin derivatives to isolated lamin B as deteted by affinity hromatography. For details see ref. 11. Lanes: 1, bound '"-labeled T-desmin; 2, olumn flow-through (not bound); 3, bound '"-labeled L-desmin; 4, olumn flow-through.

4 ell Biology: Georgatos et al. Pro. Natl. Aad. Si. USA 84 (1987) 6783 Stain Td BLOT Ld Vm i " r *T * _ Ab Ab (sp) (a) Ld 3. W J. 446 w up r.-m _ 4-4, ar, or Sp _ ~ - 7r FG. 3. M A B M G B M M G Detetion of desmin- and vimentin-binding polypeptides by an overlay assay. solated rat liver lamins A and (lane A/), lamin B (lanes B), nulear envelopes (lanes ), plasma membranes (lanes M), or whole erythroyte membrane "ghosts" (lanes G) were eletrophoresed and blotted as desribed in Materials and Methods. The blots were then inubated with iodinated T-desmin (61, pm/,g, group Td), iodinated L-desmin (48, pm/ag, group Ld), or iodinated vimentin (89, pm/&g, group Vm). The positions of lamin B (open arrowheads), vimentin (solid arrowheads), and ankyrin (solid irles) are indiated. The bands seen below vimentin represent some of its degradation produts that are also deteted by speifi antibodies. On the left, oomassie blue-stained gels of plasma membranes (M) and nulear envelopes () are depited. Solid arrowheads indiate (from top to bottom) the positions oferythroyte ankyrin, lamin A, lamin B, vimentin, and atin. lope frations. Lamin A, or other onstitutive proteins of the nulear envelopes, did not reat with these traers. Vimentin and L-desmin, but not T-desmin, bound to a band omigrating with turkey erythroyte ankyrin in plasma membrane blots (Fig. 3, lanes M). L- and T-desmin were able to bind to isolated rat liver lamin B (Fig. 3, lanes B), as well as to isolated bovine lens vimentin (data not shown). Spetrin, atin, and proteins 4.1 and 3, all present in the plasma membrane frations, did not assoiate with any of the probes (ompare staining pattern and blot patterns in Fig. 3). We notied also that the iodinated probes reated with a minor band slightly heavier than lamin B (for example, lanes ) that did not orrespond to lamin A, as judged by immunoblotting. The same minor omponent an be deteted in ertain frations of purified rat liver lamin B (Fig. 1, lane 7) and in immunoblots of rat nulear envelopes (2). Therefore, this polypeptide may represent a minor isotype of lamin B that also possesses a vimentin-binding ativity. To ensure that the deteted reativities were not due to the removal of other, perhaps more interesting, "reeptors" during subellular frationation, we tested whole "ghost" preparations from whih no proteins other than the soluble ytoplasmi ones had been removed. As seen in Fig. 3 (lanes G), intat vimentin was able to reognize ankyrin, lamin B, and endogenous vimentin, while T-desmin bound only to the latter two. No additional reativities were deteted in the ghost preparations. To show that the desmin fragments were reognizing exatly the same proteins that onstituted the vimentin reeptors, we employed immunohemial approahes in ombination with our overlay assays. When blots ontaining plasma membrane frations were tested, a polylonal antiankyrin antibody reognized the same band that reated with L-desmin, while an antibody against the a subunit of erythroid spetrin rossreated with a polypeptide migrating below it (Pig. 4). Thus we were unable to onfirm, at least by this tehnique, a previous report of a diret desmin-spetrin assoiation (26).. FG. 4. Binding of anti-a-spetrin [lane Ab-(sp)], anti-ankyrin [lane Ab-(a)], or L-desmin (lane Ld) to plasma membrane preparations. The positions of a spetrin (sp) and ankyrin (a) are indiated by arrowheads. mmunoblotting was done as desribed (11), and the solid-phase binding assay with L-desmin was done as in Materials and Methods. Finally, when urea-extrated proteins from nulear envelopes (frationated by ion-exhange hromatography and PAG; Fig. 5, DA) were tested, using anti-lamin B antibodies (Anti-B), it was found that, like exogenous vimentin, L- and T-desmin reognized always a band that orresponded to the lamin B antigen (Fig. 5, Vm, Td, and Ld). Variable binding to endogenous vimentin (oeluted with lamin B under these onditions) was also deteted, but no binding to lamin A was apparent. n sharp ontrast, when the radiolabeled middle domain of desmin was applied to the same frations there was no detetable binding (Fig. 5, Rd). DSUSSON Applying several biohemial riteria, we have shown that the F protein desmin, whih is speifially expressed in myogeni ells, interats with the same membrane reeptors as vimentin, another F subunit, expressed only in mesenhyma-derived tissues (27, 28). We dedued that desmin and vimentin (11) utilize analogous domains to assoiate with the same nulear-envelope and plasma-membrane attahment sites beause (i) elimination of the N- terminal head domains abolishes binding to the plasma membrane in both ases; (it) the middle domains do not possess any binding potential; (iii) fragments ontaining the appropriate domains but missing other parts of the moleule retain their harateristi funtional properties (that is, reognition of lamin B and ankyrin, respetively); (iv) purified vimentin displaes the desmin derivatives from the nulear envelope; and (v) diret protein-protein assoiations of desmin and vimentin with the same membrane omponents an be demonstrated in vitro. Using a solid-phase overlay assay, we obtained also some hints onerning the desmin-vimentin interations: not only did desmin and vimentin seem able to bind to eah other; in addition, they appeared to interat in a site-speifi fashion. For example, after leavage of vimentin with 2-nitro-5-thioyanobenzoi aid (8), it was found that only its N-terminal peptide assoiated with T-desmin (S.G., unpublished data). Therefore, in the desmin-vimentin "hybrid," some type of site-speifi interations must be important. Furthermore, leavage of the lamin B moleule at ysteine residues abolished vimentin and desmin binding to all of the lamin B fragments larger than 15 kda, as previously desribed (11). The onservation of suh binding ativities aross speies (birds, mammals) and aross tissues (musle, erythroytes)

5 6784 ell Biology: Georgatos et al. A- By- B.- - DA.~~~~~ Vm w_ Anti-B Td Ld Rd FG. 5. Binding of desmin fragments and vimentin to frationated polypeptides obtained from urea extrats ofnulear envelopes. Nulear envelopes were prepared, extrated with 8 M urea, and frationated by ion-exhange hromatography (11). Panel DA shows the eletrophoreti, profile of the oomassie blue-stained polypeptides in the various frations as indiated below the lanes. Panel Anti-B shows an autoradiogram of an immunoblot ofthe indiated frations with a lamin B-speifi antibody. Panels Vm, Td, Ld, and Rd represent autoradiograms of blots probed with labeled vimentin, T-desmin, L-desmin, and the desmin rod domain, respetively. Markers at left show positions of lamins A and B and of vimentin (V) ontained in eah olumn fration. The bands omigrating with lamin B in frations (panel DA) are degradation produts of lamin A, as indiated by immunoblotting (data not shown). suggests that the funtional polarity of the filament subunits must be of fundamental importane for the ellular physiology. At this point it is not lear that the funtional polarity in F-membrane interations implies neessarily a strutural polarity of the filaments per se. However, a polar arrangement of the filamentous matrix might be important for its funtion if F were used as "traks" for transporting ribonuleoprotein partiles or proteins from the nuleus to the ell surfae and vie versa. However, suh a general role of this system would onflit with the observation that several ultured ell lines an grow normally without apparently expressing any of the known types of F (29-31). The different affinities that were deteted in the assoiations of erythroyte lamin B with the desmin and vimentin moleules may imply the ourrene of a distint lamin isotype in musle ells that is more speialized in reognizing desmin rather than vimentin. Morphologial studies on striated musle ells (32, 33) revealed a lose spatial assoiation of Z-dis 1-nm filaments with some foal areas on the nulear surfae where salloping of the nulear ontour is observed together with a greater onentration of nulear Pro. Nad. Aad. Si. USA 84 (1987) pores. On the other hand, ells that oexpress desmin and vimentin, as for example BHK-21 ells, possess perinulear F omposed of both desmin and vimentin (19, 28). Therefore, it is not unreasonable to hypothesize that the binding of desmin to lamin B is of physiologial signifiane. n fat, in vitro myogenesis may offer a unique model system to study the oupling of the F with the nuleus, sine immunohemial studies have shown that whereas vimentin and desmin are oexpressed before myoblast fusion, little (if any) vimentin is present in mature musle fibers (28, 33). Therefore, it would be very interesting to examine the lamin B repertoire of these ells during myogenesis and find out if the swithing in F expression relates with a swithing in lamin expression. Suh a possibility applies, of ourse, to other ell types and in partiular to neurons, where the expression of neurofilament omponents is developmentally regulated (18). Note Added in Proof. We have reently synthesized a desmin peptide extending from the end of the rod domain (residue 415) to residue 444. 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